I generated stable HEK lines, but some lines tend to clump (others not), and when I try to separate the cells by pipetting, lots of them dye. As I intend to run flow cytometry, I guess cell climping is not very good.
Can I add some reagent to prevent this phenomenon?
Hi Philippe..first...your PBS is Ca/Mg free? because as you probably know, cell aggregation is due mainly to Ca and Mg. Also you can try to:1-wash 3X in PBS, 2-Add PBS+10uM EDTA and pipetting slowly...and then again wash in PBS...you should separate them so...
I agree with Fulvio. HEK293s are very sensitive to EDTA treatment, and this works better for flow cytometry experiments than trypsinization (better viability, no issues with neutralizing the trypsin). You'll need to incubate for longer times (10-20 minutes), but they will detach. As long as you avoid air bubbles during pipetting, the clumps should disperse with minimal cell death.
Thanks all for your advices.
I realized I used a PBS with Ca/Mg to wash the cells (before detaching them with PBS/EDTA), so I will use the correct PBS! And I will also pay attention to not pass them fully confluent (but again clumping is very cell line dependant).
Cell strainer seems to be a good device. A naive question: which pore size should I use? The cells have to flow through the strainer just by gravity?
Thanks again for your valuable answers.
very good queries and answers.. I am not using PBS for washing the cell. Washing is done with Media itself and then trypsinization with 0.5 ml (0.05% Trypsin-EDTA) by mixing and taking trypsin and incubation for 30 secs inside hood. Then after tapping.. and adding media and mixing with smooth pipetting avoiding bubble formation..These methods are good for transfection procedure.. For Flowcytometer.. cell strainer can be used.. Thank you..
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